This invention relates to genes encoding fusogenic viral membrane glycoproteins and cells expressing such genes.
Prior art methods of treating cell proliferative disorders such as cancer have involved introduction into a patient of genes or vehicles containing genes encoding, for example, proteins that enhance the immunogenicity of tumor cells. These include pro-inflammatory cytokines, T cell co-stimulators and foreign MHC proteins which produce a local bystander effect due to local inflammatory response. The local inflammatory response is said to create a cytokine-rich environment which favors the generation of a systemic bystander effect by recruitment and activation of tumor-specific T cells.
Alternatively, it has been suggested to deliver to a tumor genes encoding enzymes that render tumor cells susceptible to a xe2x80x9cpro-drugxe2x80x9d. For thymidine kinase gene transfer, there is some evidence for a local bystander effect due to transfer of ganciclovir triphosphate (the activated drug) through tight junctions to adjacent tumor cells. However, many tumors lack the requisite tight junctions and the observed local and systemic bystander effects are therefore presumed to arise because of a local inflammatory response to cells that are killed by the pro-drug with associated activation of tumor-reactive T cells.
Replicating viruses have been used extensively as oncolytic agents for experimental cancer therapy (Russell, 1994, Semin. Cancer Biol. 5, 437-443). For example, a tissue culture suspension of mumps virus was used to treat 90 patients with terminal malignancies by local application to the tumor surface, by intratumoral, oral, rectal or intravenous inoculation, or by inhalation (Asada, 1974, Cancer, 34, 1907-1928). Toxicity was minimal and in 37 of the 90 patients the tumor disappeared or decreased to less than half of its initial size. Minor responses were observed in a further 42 patients. Tumor destruction was maximal several days after virus administration and was often followed by long-term suppression of tumor growth, perhaps due to stimulation of antitumor immunity.
Other viruses that have been used for cancer therapy in human subjects or experimental mouse models include West Nile virus, herpes simplex virus, Russian Far East encephalitis, Newcastle disease virus, Venezuelan equine encephalomyelitis, rabies, vaccinia and varicella (Russell, 1994, Eur. J. Cancer, 30A, 1165-1171). The rationale for these studies has been that many viruses replicate and spread more rapidly in neoplastic tissues than in nontransformed tissues and might therefore be expected to cause more damage to the tumor than to the host.
It is an object of the invention to provide compositions and methods for selective elimination of unwanted cells.
Another object of the invention is to selectively eliminate target cells by achieving a bystander effect.
Another object of the invention is to selectively induce syncytium formation of target cells, thereby eliminating the target cells.
The invention encompasses compositions comprising pharmaceutical formulations comprising a recombinant nucleic acid vector comprising a nucleotide sequence encoding a syncytium-inducing polypeptide expressible on a eukaryotic cell surface in admixture with a pharmaceutically acceptable carrier.
The invention also encompasses compositions comprising pharmaceutical formulations comprising a eukaryotic host cell containing a recombinant nucleic acid vector comprising a nucleotide sequence encoding a syncytium-inducing polypeptide and expressing the polypeptide on its surface, in admixture with a pharmaceutically acceptable carrier.
Preferably, in a composition according to the invention the sequence encodes at least a fusogenic portion of a viral fusogenic membrane glycoprotein.
Preferably, the sequence encodes a non-naturally occurring polypeptide. xe2x80x9cNon-naturally occurring polypeptide refers to a recombinant polypeptide; for example, a chimeric polypeptide.
Preferably, the sequence encodes a fusogenic membrane glycoprotein having an artificially introduced protease-cleavage site.
Preferably, the sequence encodes a fusogenic membrane glycoprotein having an altered binding specificity.
Preferably, the sequence encodes a fusogenic membrane glycoprotein having enhanced fusogenicity, for example, as results from truncation of the carboxy terminal portion of a fusogenic membrane glycoprotein.
The eukaryotic host cell may be a human cell, such as a host cell selected from the group consisting of neoplastic cells, migratory cells, T lymohocytes, B lymphocytes or other haemopoietic cells.
The invention also features a method of eliminating unwanted cells of a cell proliferative disease in a human patient, comprising administering to the patient a pharmaceutical formulation according to the invention in an amount sufficient to cause fusion of those cells which cause the cell proliferative disease.
The invention also encompasses kits comprising a pharmaceutical formulation described herein, and packaging means therefore.
Nucleic acid vectors and host cells of the invention are useful in gene therapy of diseases involving cell proliferative disorders, where it is desired that cells which proliferate undesirably or uncontrollably are selectively eliminated. Such diseases include but are not limited to malignant diseases. The vector encoding the syncytium-inducing polypeptide or a host cell expressing on its surface a syncytium-inducing polypeptide is administered to an affected individual so as to cause cell-cell fusion of unwanted cells.
Preferably, the syncytium-inducing polypeptide comprises at least a fusogenic portion of a viral fusogenic membrane glycoprotein (which may be abbreviated as FMG). In some embodiments, it is preferred that the syncytium-inducing polypeptide is capable of inducing syncytium formation at substantially neutral pH (i.e. pH 6-8). Many suitable FMGs will be known to those skilled in the art and several are provided hereinbelow.
Typically the vector will be adapted so as to express the syncytium-inducing polypeptide on the surface of a human cell, such that, when properly expressed, the polypeptide may cause the cell to fuse with other human cells which do not express the syncytium-inducing polypeptide.
It is preferred that, where the polypeptide comprises a viral FMG, the FMG is expressed in substantial isolation from other viral components and thus consists essentially of those viral components which are essential for fusogenic activity on target cells (e.g. where two viral glycoproteins are required for syncytium formation, such as the xe2x80x98Fxe2x80x99 and xe2x80x98Hxe2x80x99 glycoproteins of Paramyxoviridae both being required for syncytium-formation).
In addition, it will frequently be desirable to xe2x80x9cengineerxe2x80x9d the syncytium-inducing polypeptide to optimize its characteristics for therapeutic use, such that the vector directs the expression of a xe2x80x9cnon-naturally occurringxe2x80x9d polypeptide.
Preferred modifications include truncation of the cytoplasmic domain of a glycoprotein so as to increase its fusiongenic activity; introduction of novel binding specificities or protease-dependencies into fusogenic viral membrane glycoproteins and thereby to target their fusogenic activities to specific cell types that express the targeted receptors or to specific microenvironments that are rich in the appropriate activating proteases.
The invention provides a method of treating a cell proliferative disease such as a malignant disease in a human patient, comprising administering to the patient a recombinant nucleic acid directing the expression of a syncytium inducing polypeptide in a human cell, such that cells (xe2x80x9cindexxe2x80x9d cells) of the patient which take up the recombinant nucleic acid will fuse with the proliferating cells, e.g., cancerous cells (xe2x80x9ctargetxe2x80x9d cells) causing the disease.
In a particular embodiment, the nucleic acid is introduced in vitro into suitable human index cells (by any one of various known standard techniques, such as transfection, transduction or transformation), and the index cells are then introduced into the patient, where they can exert a syncytium-inducing effect on target cells.
The invention also provides for use of a recombinant nucleic acid vector in the gene therapy of a cell proliferative disorder such as a malignant disease, the vector comprising a sequence directing the expression on a eukaryotic cell surface of a syncytium-inducing polypeptide.
The invention also provides a recombinant nucleic acid vector for use in the preparation of a medicament to treat a cell proliferative disease such as a malignant disease in a human patient, the vector comprising a sequence directing the expression on a eukaryotic cell surface of a syncytium-inducing polypeptide.
The invention also provides a host cell comprising a recombinant nucleic acid vector in accordance with the invention defined above. The cell will typically be a eukaryotic cell (especially a human cell) and desirably will express on its surface a syncytium-inducing polypeptide.
As used herein, the term xe2x80x9csyncytium inducing polypeptidexe2x80x9d refers to a polypeptide or a portion thereof that induces cell-cell fusion resulting in formation of a syncytium.
The term xe2x80x9csyncytiumxe2x80x9d refers to a cell-cell fusion which appears in a tissue biopsy or tissue culture sample as a large acellular area with multiple nucleii, i.e., a multinucleate region of cytoplasm.
xe2x80x9cEnhanced induction of syncytium formationxe2x80x9d refers to the biological activity of a syncytium inducing polypeptide in which the enhancement is an increase in the number of cells that are induced to form a syncytium above (at least 10-20%) the level of that observed without the syncytium inducing polypeptide or, if the syncytium inducing polypeptide is engineered to achieve the enhanced activity, then above the level of that observed using the non-engineered polypeptide. xe2x80x9cEnhanced fusogenic activityxe2x80x9d is also used herein to refer to enhanced syncytium inducing activity.
xe2x80x9cNonviable syncytiumxe2x80x9d refers to syncytium that do not survive for longer than 48-72 hours in tissue culture (i.e., in vitro), or a syncytium which is immunogenic (recognized by the immune system) in vivo and are nonviable in an immunocompetent host.
As used herein, the term xe2x80x9csubstantial isolationxe2x80x9d of a viral polypeptide or gene encoding a viral polypeptide, with respect to other viral components, means that most of the other components of the virus (those not necessary for fusogenic activity of the virus polypeptide) are absent, and thus the DNA or viral polypeptide consists essentially of those viral components which are essential for fusogenic activity on target cells.
A xe2x80x9cfusogenic effectxe2x80x9d refers to the natural biological activity of a fusogenic polypeptide in inducing cell fusion via the presence of a virus encoding and expressing the fusogenic polypeptide. Virus-cell fusion and cell-cell fusion are distinct processes. xe2x80x9cFusogenicxe2x80x9d refers to the biological activity of a viral membrane glycoprotein to promote virus-cell fusion when in its natural virus context. In contrast, xe2x80x9csyncytium-inductionxe2x80x9d refers to the biological activity of a syncytium-inducing polypeptide, which may be a viral membrane glycoprotein substantially isolated from its natural virus context, to induce cell-cell fusion without the virus. To be useful according to the invention, a viral glycoprotein which has a fusogenic effect when carried in the virus must be capable of inducing syncytium formation when in substantial isolation from the virus.
A xe2x80x9cfusogenic portionxe2x80x9d refers to a portion of a fusogenic virus membrane polypeptide which possesses fusogenic activity and thus promotes virus-cell fusion.
xe2x80x9cAltered receptor specificityxe2x80x9d refers to a modification in a ligand such that the receptor recognized by the modified ligand is altered from a first receptor to a second receptor; that is, the unmodified ligand recognizes a first receptor and the modified ligand recognizes a second receptor.
xe2x80x9cNovel protease-dependencyxe2x80x9d of a polypeptide according to the invention refers to the presence of a new protease sensitive site that is susceptible to cleavage where a site of proteolysis is artificially introduced into a given protein, and the protein containing the new sensitivity is dependent for becoming biologically active upon a protease that specifically cleaves the protein at the site of proteolysis; without cleavage by the protease at the new protease sensitive site, the protease-dependent polypeptide will not become biologically active.
A xe2x80x9cvector systemxe2x80x9d refers to one vector or several vectors which together encode specified components.
The invention will now be further described by way of illustrative example and with reference to the accompanying drawing, FIG. 1, which is a schematic representation of a recombinant nucleic acid vector in accordance with the invention.